Insulin provides a model for analysis of protein folding and recognition with application to human therapeutics. During the previous grant period novel misfolded states ( kinetic traps ) and protein-folding intermediates have been characterized. In this Competing Application we propose to combine combinatorial peptide chemistry and NMR spectroscopy to define structural determinants of foldability and function. The results promise to provide general insight into mechanisms of protein folding with application to the design of analogs of clinical interest. Experiments will test the following propositions: Hypothesis 1. As crystal structures represent inactive conformers, the hormone reorganizes on receptor binding; Hypothesis 2. The native insulin sequence represents an evolutionary compromise between competing constraints of foldability and function; and Hypothesis 3. Distinct structural determinants of foldability and function may be resolved by combinatorial peptide chemistry in vitro and combinatorial mutagenesis in vivo. As an assay of foldability, chain combination and mass spectrometry will be used to distinguish between allowed and disallowed sequence variants in a random peptide library. Representative members of such libraries will be tested for binding to the insulin receptor and will be characterized by NMR. To circumvent self-association, analogs will be constructed in the context of a monomeric template ( DKP-insulin ). Collaborative in vivo analysis of prohormone folding will be conducted by Prof. D. F. Steiner. By thus combining NMR spectroscopy with modern methods of combinatorial chemical synthesis, this application offers the exciting possibility of delineating structural mechanisms of specific disulfide pairing in a protein. Moreover, correlation of enhanced (or decreased) receptor binding with sites of structural change (or conservation) will define mechanisms of receptor recognition. This information is of basic importance in protein chemistry and likely to facilitate design of novel insulin for the treatment of diabetes mellitus.